Proteins and especially immunoglobulins play an important role in today's medical portfolio. Polypeptides for use in pharmaceutical applications are mainly produced in mammalian cells such as CHO cells, NS0 cells, Sp2/0 cells, COS cells, HEK cells, BHK cells, PER.C6® cells, and the like.
Due to their chemical and physical properties, such as molecular weight and domain architecture including secondary modifications, the downstream processing of immunoglobulins is very complicated. For example, are not only for formulated drugs but also for intermediates in downstream processing (DSP) concentrated solutions required to achieve low volumes for economic handling and application storage. The downstream processing of biotechnologically produced immunoglobulins typically comprises three chromatography steps: a first affinity chromatography step using e.g. Protein A, to remove non-immunoglobulin molecules, followed by two ion exchange chromatography steps. The purified immunoglobulin is obtained in a low concentration solution requiring a concentration step prior to formulating the antibody into the pharmaceutical formulation. Many proteins, including IgG, form dimers, oligomers or higher order aggregates. In order to provide a therapeutic protein product with the required purity these molecule species have to be removed by the purification process.
Different methods are well established and widely used for protein purification (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75 (1998) 93-102). Zhou, J. X., et al. (J. Chrom. A 1175 (2007) 69-80) report a pH-conductivity hybrid gradient cation-exchange chromatography for process-scale monoclonal antibody purification. The separation of antibody monomers from its multimers by use of ion exchange chromatography is reported in EP 1 084 136. U.S. Pat. No. 5,429,746 relates to the application of hydrophobic interaction chromatography combination chromatography to the purification of antibody molecule proteins.
Solvent modulation of column chromatography is reported by Arakawa, T., et al. in Prot. Pep. Lett. 15 (2008) 544-555. In EP 1 729 867 a method for chromatographic purification is reported. Antibody aggregate removal by hydroxyapatite chromatography is reported by Gagnon, P. and Beam, K., in Curr. Pharm. Biotechnol. 10 (2009) 440-446. In J. Immunol. Meth. (336 (2008) 222-228) reports Gagnon, P., an improved antibody aggregate removal by hydroxyapatite chromatography in the presence of polyethylene glycol. In WO 2004/013162 an increased dynamic binding capacity in ion exchange chromatography by addition of polyethylene glycol is reported. In WO 2005/094960 a method for chromatographic purification is reported. Enhanced capacity and purification of antibodies by mixed mode chromatography in the presence of aqueous-soluble non-ionic organic polymers is reported in WO 2008/086335. In WO 2009/149067 a process for the purification of antibodies is reported.
Milby, K. H., et al. report the ion-exchange chromatography of proteins and the effect of neutral polymers in the mobile phase (J. Chrom. 482 (1989) 133-144). In Biotechnology Techniques (12 (1998) 289-293) Feng, X-L., et al. report that polyethylene glycol improves the purification of recombinant human tumor necrosis factor during ion exchange chromatography. A method for obtaining unique selectivities in ion-exchange chromatography by addition of organic polymers to the mobile phase is reported by Gagnon, P., et al. (J. Chrom. 743 (1996) 51-55). Gagnon, P., reports in an internet article on validated.com (http://www.validated.com/revalbio/pdffiles/ionslect.pdf) the fine-tuning of selectivity on ion exchangers. In WO 2009/149067 a process for the purification of antibodies is reported.